DMD Mutations Research Articles

MLPA followed by target-NGS to detect mutations in the dystrophin gene of Peruvian patients suspected of DMD/DMB.

BackgroundWe report the molecular analysis of the DMD gene in a group of Peruvian patients with Duchenne/Becker dystrophinopathy. This is the first study to thoroughly characterize mutations in this population.MethodsWe used the combination of multiplex ligation‐dependent probe amplification (MLPA) and sequencing analysis of the DMD gene. We recruited Peruvian patients in 2 years from reference national hospitals. We performed DNA tests in 152 patients, checking first exon deletion/duplication by MLPA, and subsequently, if negative, samples were sequenced to detect point mutations.ResultsThe average age for diagnosis was 9.8 years, suggesting a delay for timely diagnosis and care. We found causal DMD mutations in 125 patients: 72 (57.6%) exon deletions/duplications (41.6% deletions, 16.0% duplications), and 53 (42.4%) point mutations (27.2% nonsense, 9.6% small indels, and 5.6% splice site).ConclusionDue to our genetic background, we expected a higher number of novel and recurrent causal mutations in our sample. Results showed 16% of novel mutations, similar to other well‐studied populations.

Murine models of Duchenne muscular dystrophy: is there a best model?

Murine models of Duchenne muscular dystrophy: is there a best model?

Focus on the road to modelling cardiomyopathy in muscular dystrophy.

Alterations in the DMD gene, which codes for the protein dystrophin, cause forms of dystrophinopathies such as Duchenne muscular dystrophy, an X-linked disease. Cardiomyopathy linked to DMD mutations is becoming the leading cause of death in patients with dystrophinopathy. Since phenotypic pathophysiological mechanisms are not fully understood, the improvement and development of new disease models, considering their relative advantages and disadvantages, is essential. The application of genetic engineering approaches on induced pluripotent stem cells, such as gene-editing technology, enables the development of physiologically relevant human cell models for in vitro dystrophinopathy studies. The combination of induced pluripotent stem cells-derived cardiovascular cell types and 3D bioprinting technologies hold great promise for the study of dystrophin-linked cardiomyopathy. This combined approach enables the assessment of responses to physical or chemical stimuli, and the influence of pharmaceutical approaches. The critical objective of in vitro microphysiological systems is to more accurately reproduce the microenvironment observed in vivo. Ground-breaking methodology involving the connection of multiple microphysiological systems comprised of different tissues would represent a move toward precision body-on-chip disease modelling could lead to a critical expansion in what is known about inter-organ responses to disease and novel therapies that have the potential to replace animal models. In this review, we will focus on the generation, development, and application of current cellular, animal, and potential for bio-printed models, in the study of the pathophysiological mechanisms underlying dystrophin-linked cardiomyopathy in the direction of personalized medicine.

Prevalence and clinical outcomes of dystrophin-associated dilated cardiomyopathy without severe skeletal myopathy.

Dilated cardiomyopathy (DCM) associated with dystrophin gene (DMD) mutations in individuals with mild or absent skeletal myopathy is often indistinguishable from other DCM forms. We sought to describe the phenotype and prognosis of DMD associated DCM in DMD mutation carriers without severe skeletal myopathy. At 26 European centres, we retrospectively collected clinical characteristics and outcomes of 223 DMD mutation carriers (83% male, 33 ± 15 years). A total of 112 individuals (52%) had DCM at first evaluation [n= 85; left ventricular ejection fraction (LVEF) 34 ± 11.2%] or developed DCM (n= 27; LVEF 41.3± 7.5%) after a median follow-up of 96 months (interquartile range 5-311 months). DCM penetrance was 45% in carriers older than 40 years. DCM appeared earlier in males and was independent of the type of mutation, presence of skeletal myopathy, or elevated serum creatine kinase levels. Major adverse cardiac events (MACE) occurred in 22% individuals with DCM, 18% developed end-stage heart failure and 9% sudden cardiac death or equivalent. Skeletal myopathy was not associated with survival free of MACE in patients with DCM. Decreased LVEF and increased left ventricular end-diastolic diameter at baseline were associated with MACE. Individuals without DCM had favourable prognosis without MACE or death during follow-up. DMD-associated DCM without severe skeletal myopathy is characterized by incomplete penetrance but high risk of MACE, including progression to end-stage heart failure and ventricular arrhythmias. DCM onset is the major determinant of prognosis with similar survival regardless of the presence of skeletal myopathy.

Matrilineal analysis of mutations in the DMD gene in a multigenerational South Indian cohort using DMD gene panel sequencing.

BackgroundDuchenne muscular dystrophy (DMD) is an X‐linked recessive neuromuscular disorder characterised by progressive irreversible muscle weakness, primarily of the skeletal and the cardiac muscles. DMD is characterised by mutations in the dystrophin gene, resulting in the absence or sparse quantities of dystrophin protein. A precise and timely molecular detection of DMD mutations encourages interventions such as carrier genetic counselling and in undertaking therapeutic measures for the DMD patients.ResultsIn this study, we developed a 2.1 Mb custom DMD gene panel that spans the entire DMD gene, including the exons and introns. The panel also includes the probes against 80 additional genes known to be mutated in other muscular dystrophies. This custom DMD gene panel was used to identify single nucleotide variants (SNVs) and large deletions with precise breakpoints in 77 samples that included 24 DMD patients and their matrilineage across four generations. We used this panel to evaluate the inheritance pattern of DMD mutations in maternal subjects representing 24 DMD patients.ConclusionHere we report our observations on the inheritance pattern of DMD gene mutations in matrilineage samples across four generations. Additionally, our data suggest that the DMD gene panel designed by us can be routinely used as a single genetic test to identify all DMD gene variants in DMD patients and the carrier mothers.

Duchenne muscular dystrophy.

Duchenne muscular dystrophy is a severe, progressive, muscle-wasting disease that leads to difficulties with movement and, eventually, to the need for assisted ventilation and premature death. The disease is caused by mutations in DMD (encoding dystrophin) that abolish the production of dystrophin in muscle. Muscles without dystrophin are more sensitive to damage, resulting in progressive loss of muscle tissue and function, in addition to cardiomyopathy. Recent studies have greatly deepened our understanding of the primary and secondary pathogenetic mechanisms. Guidelines for the multidisciplinary care for Duchenne muscular dystrophy that address obtaining a genetic diagnosis and managing the various aspects of the disease have been established. In addition, a number of therapies that aim to restore the missing dystrophin protein or address secondary pathology have received regulatory approval and many others are in clinical development.

Ethnicity-related DMD Genotype Landscapes in European and Non-European Countries.

ObjectiveGenetic diagnosis and mutation identification are now compulsory for Duchenne (DMD) and Becker muscular dystrophies (BMD), which are due to dystrophin (DMD) gene mutations, either for disease prevention or personalized therapies. To evaluate the ethnic-related genetic assortments of DMD mutations, which may impact on DMD genetic diagnosis pipelines, we studied 328 patients with DMD and BMD from non-European countries.MethodsWe performed a full DMD mutation detection in 328 patients from 10 Eastern European countries (Poland, Hungary, Lithuania, Romania, Serbia, Croatia, Bosnia, Bulgaria, Ukraine, and Russia) and 2 non-European countries (Cyprus and Algeria). We used both conventional methods (multiplex ligation-dependent probe amplification [MLPA] followed by gene-specific sequencing) and whole-exome sequencing (WES) as a pivotal study ran in 28 patients where DMD mutations were already identified by standard techniques. WES output was also interrogated for DMD gene modifiers.ResultsWe identified DMD gene mutations in 222 male patients. We identified a remarkable allele heterogeneity among different populations with a mutation landscape often country specific. We also showed that WES is effective for picking up all DMD deletions and small mutations and its adoption could allow a detection rate close to 90% of all occurring mutations. Gene modifiers haplotypes were identified with some ethnic-specific configurations.ConclusionsOur data provide unreported mutation landscapes in different countries, suggesting that ethnicity may orient genetic diagnosis flowchart, which can be adjusted depending on the mutation type frequency, with impact in drug eligibility.

Prenatal diagnosis of Duchenne muscular dystrophy revealed a novel mosaic mutation in Dystrophin gene: a case report

sBackgroundDuchenne muscular dystrophies (DMDs) are X-linked recessive neuromuscular disorders with malfunction or absence of the Dystrophin protein. Precise genetic diagnosis is critical for proper planning of patient care and treatment. In this study, we described a Chinese family with mosaic DMD mutations and discussed the best method for prenatal diagnosis and genetic counseling of X-linked familial disorders.MethodsWe investigated all variants of the whole dystrophin gene using multiple DNA samples isolated from the affected family and identified two variants of the DMD gene in a sick boy and two female carriers by targeted next generation sequencing (TNGS), Sanger sequencing, and haplotype analysis.ResultsWe identified the hemizygous mutation c.6794delG (p.G2265Efs*6) of DMD in the sick boy, which was inherited from his mother. Unexpectedly, a novel heterozygous mutation c.6796delA (p.I2266Ffs*5) of the same gene, which was considered to be a de novo variant, was detected from his younger sister instead of his mother by Sanger sequencing. However, further NGS analysis of the mother and her amniotic fluid samples revealed that the mother carried a low-level mosaic c.6796delA mutation.ConclusionsWe reported two different mutations of the DMD gene in two siblings, including the novel mutation c.6796delA (p.I2266Ffs*5) inherited from the asymptomatic mosaic-carrier mother. This finding has enriched the knowledge of the pathogenesis of DMD. If no mutation is detected in obligate carriers, the administration of intricate STR/NGS/Sanger analysis will provide new ideas on the prenatal diagnosis of DMD.

Phenotype and clinical outcomes of dystrophin associated dilated cardiomyopathy

Abstract Background Mutations in dystrophin gene (DMD) can cause skeletal myopathy and dilated cardiomyopathy (DCM) independently or in combination. Natural history of DMD mutation carriers and dystrophin-associated DCM is poorly understood. Objectives This study sought to describe phenotype and prognosis of DMD mutations in a large multicenter cohort of Non-Duchenne DMD mutations carriers. Methods The study cohort comprised 223 individuals with a DMD mutation (83% males, 33±15 years at first evaluation) followed at 26 European centers. Major adverse cardiac events (MACE) were defined as a composite of cardiac death, heart transplant, LVAD implantation, aborted SCD or appropriate ICD shock. Results At initial evaluation, 85 patients (38%) had DCM (52 in combination with muscular disease) and 92 (41%) had isolated muscular disease. After a median follow-up of 96 months, 112 individuals (53%) had DCM and 20% of the individuals who had normal cardiac function at baseline developed DCM. DCM penetrance by age 30 was 56%. DCM onset was associated with male sex and was independent of the type of mutation, the presence of skeletal myopathy or serum creatine kinase levels. MACE occurred in 11% and 22% individuals from the entire cohort and with DCM respectively, and were more frequent in DCM patients without muscular disease than in those with skeletal myopathy (35.5% vs 17.7%; p=0.04). Among patients with DCM, 18% developed end-stage heart failure and 9% a major arrhythmic event (SCD/aborted SCD/ICD shock/VT). There were not differences in survival between patients with isolated DCM and those with DCM and muscular phenotype. Decreased LVEF and increased left ventricular end-diastolic diameter at baseline were associated with MACE. Atrial fibrillation and neurological events were also frequent. Prognosis of individuals who did not develop DCM was good with 96% survival during follow-up. Conclusions DCM caused by mutations in DMD is characterized by moderate penetrance but a high risk of MACE, progression to end-stage heart failure and ventricular arrythmias. DCM onset is the major determinant of prognosis in DMD mutation carriers with similar survival irrespectively of the presence of concomitant muscular disease. Survival free of MACE analysis Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Instituto de Salud Carlos III. Contratos i-PFIS: Doctorados IIS-empresa en Ciencias y Tecnologías de la Salud

DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.218 A novel in-frame deletion of exons 52-55 DMD mouse model preserves muscle function

Duchenne muscular dystrophy (DMD) is a life-limiting X-linked recessive neuromuscular disorder, affecting approximately 1 in 5000 boys. DMD is caused by mutations in the DMD gene encoding dystrophin, a member of the dystrophin glycoprotein complex (DGC) which protects muscle fibers from contraction-induced injury. DMD is marked by absent dystrophin expression, often caused by frame-shift mutations. Among the dystrophinopathies, Becker muscular dystrophy (BMD) is a milder disorder typically caused by in-frame DMD mutations, resulting in shortened yet partly functional dystrophin formation. Both the clinical presentation and prognosis of BMD patients are improved compared to those with DMD. Therefore, conversion of out-of-frame Dmd mutations into in-frame mutations by means of exon-skipping, is appealing as a potential therapeutic option. This study first created a DMD mouse model mirroring a patient deletion of exons 52-54 (Dmd Δ52-54). Such a mutation may be amenable to exon skipping to create a deletion of exons 52-55. We then generated a mouse model of the in-frame deletion mutation Dmd Δ52-55, recapitulating the result of exon skipping in our DMD mouse. To date, Dmd Δ52-55 is the first BMD mouse model generated. Both mouse models were subsequently subjected to histopathological and functional analysis. Dmd Δ52-54 mice lacked dystrophin expression, exhibited a muscular dystrophy phenotype, and had compromised motor and cardiac function. In contrast, the Dmd Δ52-55 mice showed expression of a truncated dystrophin which localized correctly to the sarcolemma and recruited DGC components, which were absent in Dmd Δ52-54. Furthermore, this shortened dystrophin maintained muscle integrity and protected from exercise-induced damage similar to wildtype dystrophin. Thus, our results indicate that therapies targeting the conversion of DMD-causing frameshift mutations into BMD-like in-frame mutations may restore shortened dystrophin expression and improve dystrophic phenotype.

DMD – THERAPY: P.289 Open-label evaluation of eteplirsen in males with DMD amenable to exon 51 Skipping: PROMOVI

Eteplirsen received accelerated FDA approval for treatment of DMD patients with mutations amenable to exon 51 skipping. We report results from a phase 3 open-label study evaluating eteplirsen in boys with DMD amenable to exon 51 skipping therapy. Males aged 7 to 16 years inclusive, with confirmed DMD and frame-shift mutations amenable to treatment by exon 51 skipping, received intravenous (IV) eteplirsen 30 mg/kg/wk for 96 wk. An untreated cohort of boys with DMD not amenable to exon 51 skipping was also enrolled.

Molecular alterations in meningioma: prognostic and therapeutic perspectives.

To discuss recent advances in the meningioma biology and their clinical implications. Meningioma is the most common primary intracranial tumor. Mostly benign, 20% of cases display an aggressive behavior despite best standard of care. The genetic landscape of meningiomas is divided according to NF2 mutational status. Although about 60% of meningiomas display NF2 mutations, the other share is more heterogenous. Mutations in TRAF7, SMO, v-akt murine thymoma viral oncogene homolog 1 (AKT1), PI3KCA and KLF4 are seen mostly in WHO grade 1 meningiomas. In higher grade meningiomas, mutations of the TERT promoter and deletions of CDKN2A/B emerge and have prognostic value. Moreover, mutations in DMD, BAP1 and PBRM1 have recently been discovered and are being further explored. DNA methylation subgroups offer valuable insight into meningioma prognosis and its implementation in clinical setting is under evaluation. Moreover, the study of distinct meningioma populations such as radiation-induced meningioma and progestin-associated meningioma may provide further insight into meningioma oncogenesis and potential therapeutic targets. The mutational landscape of meningioma has expanded following the use of the new genetic sequencing approaches. Novel mutations have been characterized and reveal their prognostic and therapeutic applications. This improved understanding of meningioma biology has promising implications for novel treatment strategies.

Pathological evaluation of rats carrying in-frame mutations in the dystrophin gene: a new model of Becker muscular dystrophy.

ABSTRACTDystrophin, encoded by the DMD gene on the X chromosome, stabilizes the sarcolemma by linking the actin cytoskeleton with the dystrophin-glycoprotein complex (DGC). In-frame mutations in DMD cause a milder form of X-linked muscular dystrophy, called Becker muscular dystrophy (BMD), characterized by the reduced expression of truncated dystrophin. So far, no animal model with in-frame mutations in Dmd has been established. As a result, the effect of in-frame mutations on the dystrophin expression profile and disease progression of BMD remains unclear. In this study, we established a novel rat model carrying in-frame Dmd gene mutations (IF rats) and evaluated the pathology. We found that IF rats exhibited reduced expression of truncated dystrophin in a proteasome-independent manner. This abnormal dystrophin expression caused dystrophic changes in muscle tissues but did not lead to functional deficiency. We also found that the expression of additional dystrophin named dpX, which forms the DGC in the sarcolemma, was associated with the appearance of truncated dystrophin. In conclusion, the outcomes of this study contribute to the further understanding of BMD pathology and help elucidate the efficiency of dystrophin recovery treatments in Duchenne muscular dystrophy, a more severe form of X-linked muscular dystrophy.

Myopathies with finger flexor weakness: Not only inclusion-body myositis.

Muscle disorders are characterized by differential involvement of various muscle groups. Among these, weakness predominantly affecting finger flexors is an uncommon pattern, most frequently found in sporadic inclusion-body myositis. This finding is particularly significant when the full range of histopathological findings of inclusion-body myositis is not found on muscle biopsy. Prominent finger flexor weakness, however, is also observed in other myopathies. It occurs commonly in myotonic dystrophy types 1 and 2. In addition, individual reports and small case series have documented finger flexor weakness in sarcoid and amyloid myopathy, and in inherited myopathies caused by ACTA1, CRYAB, DMD, DYSF, FLNC, GAA, GNE, HNRNPDL, LAMA2, MYH7, and VCP mutations. Therefore, the finding of finger flexor weakness requires consideration of clinical, myopathological, genetic, electrodiagnostic, and sometimes muscle imaging findings to establish a diagnosis.

Pediatric clear cell meningioma involving the middle cranial fossa in the context of NF2 and SMARCE1 mutations

Meningiomas are an uncommon entity in children and adolescents. <30 cases of pediatric clear cell meningioma (CCM), a World Health Organization (WHO) Grade II tumor, have been reported in the literature. These tumors are more likely to recur than the more common WHO Grade I meningiomas, especially with incomplete surgical resection. CCMs are most commonly found in the spine and posterior cranial fossa. Recently, SMARCE1 mutations have been linked to the development of CCM. To evaluate the progression of pediatric CCM in the context of emerging genetic knowledge, we reviewed all 45 cases of CCM at our institution for a 23 year period (1997–2019) to identify pediatric cases. Forty-four of the tumors arose in adults from age 34–81 years. The one pediatric case originally presented at age 4 years; the patient was found to have a CCM in the left cavernous sinus projecting into the posterior fossa, associated with a novel germline SMARCE1 mutation and somatic NF1 and DMD mutations. After two years, the patient had a recurrence of the tumor and underwent a second resection. This is the 5th reported case of CCM in the middle cranial fossa, and the only recurrent case, as well as the only reported case of recurrent pediatric CCM associated with a germline SMARCE1 mutation. Further study of the natural history of tumors associated with germline SMARCE1 loss could potentially inform prognosis.

Abstract B026: The genomic landscape of canine osteosarcoma implicates DMD as a therapeutic vulnerability

Abstract Background: Osteosarcoma (OS), the most common malignant bone tumor in humans and dogs, shares several features in both species including clinical presentation and molecular alterations. Despite numerous efforts there have been no improvements in outcome: 30% of people and 90% of dogs still die of metastasis. While the genomic landscape of human OS has been interrogated, limited data exist regarding that of dogs. Using whole genome sequencing (WGS) we identified the presence of mutations in DMD, the gene encoding dystrophin, in canine OS. The purpose of this work was to expand upon these findings and begin to interrogate the functional consequences of these mutations. Methods: Whole-genome sequencing (WGS) of 24 canine primary OS tumor samples was completed on an Illumina platform. Somatic single nucleotide and structural variants, and germline variants were identified using Cerberus, a canine cancer genomics pipeline. Expression of the Dp71 DMD isoform was modulated using CRISPR-Cas9 and lentiviral overexpression approaches; the biologic significance of DMD loss was characterized using Co-IP, western blotting, Matrigel invasion and proliferation assays. Results: Our prior data found that canine OS recapitulates several genetic features of human OS including high structural complexity and translocation burden withTP53 (71%) the most frequently mutated gene. In addition to inactivating SETD2 mutations 42% of cases, we found that 50% of tumors possessed large somatic intragenic deletions and copy number loss of DMD, the gene encoding dystrophin. Building upon these data we found that truncated dystrophin isoforms (Dp260, Dp140, Dp71, Dp40) predominate in canine OS cell lines, with only Dp40 expressed in normal osteoblasts. Deletion of Dp71 in OS lines using CRISPR-Cas9 resulted in increased cell proliferation and markedly enhanced invasion through Matrigel. Consistent with the published literature, dystrophin and β-dystroglycan co-associated in the OS lines, and phosphorylation of β-dystroglycan was increased when dystrophin expression was downregulated. Lastly, dasatinib, a small molecule inhibitor of Src, inhibited β-dystroglycan phosphorylation and cell proliferation in the canine OS lines. Conclusions: These data provide important new information regarding the genomic profile of canine OS and suggest that aberrations in dystrophin expression may contribute to OS pathophysiology in dogs by promoting a metastatic phenotype. Future work will dissect exactly how altered dystrophin isoforms contribute to OS tumor biology with the goal of developing approaches for therapeutic intervention that target this pathway. Citation Format: Heather L. Gardner, Karthigayini Sivaprakasam, Natalia Briones, William Hendricks, Cheryl A. London. The genomic landscape of canine osteosarcoma implicates DMD as a therapeutic vulnerability [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B026. doi:10.1158/1535-7163.TARG-19-B026

Relationships between DMD mutations and neurodevelopment in dystrophinopathy.

We performed a prospective, cross-sectional analysis of neurodevelopmental concerns and psychosocial adjustment in relation to DMD mutations in young steroid-naive boys with dystrophinopathy. We evaluated 196 steroid-naive boys with dystrophinopathy who were enrolled in the Finding the Optimal Regimen for Duchenne Muscular Dystrophy trial. The neurodevelopmental concerns and psychosocial adjustment challenges were analyzed in relation to DMD mutation. A parent or legal guardian reported neurodevelopmental concerns in 4 domains (speech, learning and attentional difficulties, and autism spectrum disorder [ASD]) and completed the Personal Adjustment and Role Skills Scale to assess psychosocial adjustment. We also assessed whether boys of DMD carrier mothers were more vulnerable to speech delay and learning difficulties. We found that 39% of boys were reported to have speech delay with a mean age of speaking at 28 months (range 7-66 months). Learning difficulties were reported in 28% of participants. Inattentive-overactive and oppositional-defiant behavior was reported in 8% and 5% of participants, respectively. Psychosocial adjustment challenges were reported in 4% of participants. An ASD diagnosis was reported in 3 participants. Speech delay and learning difficulties were more common in boys with mutations downstream of DMD exon 45. Neurodevelopmental concerns were not associated with DMD deletion, duplication, or point mutation subtype. Boys of DMD carrier mothers did not have longer speech delay or more learning difficulties. Our data support evidence for a relationship between neurodevelopmental concerns and DMD mutation. A longitudinal assessment of developmental trajectory is necessary to evaluate how specific DMD mutations affect brain function.

P.142Very low residual dystrophin levels mitigate dystrophinopathy towards Becker muscular dystrophy

Novel therapy approaches for Duchenne muscular dystrophy aim at restoring dystrophin expression. However, the minimal dystrophin restoration level required to achieve clinical benefit remains unknown. Residual dystrophin expression can be observed in certain patients carrying DMD intronic (including splice sites) and nonsense mutations in exons for which deletion leads to in-frame transcripts. To determine whether expression of very low dystrophin levels (<5%) can mitigate disease severity, residual dystrophin expression was correlated with disease severity of patients with these DMD mutations. Seventy-seven patients were identified using the UMD-DMD-France database. Clinical data were obtained from medical care centres. Residual dystrophin expression was semi-quantitatively determined on Western Blots. Time-to-event analysis was performed for age of loss-of-ambulation as well as for respiratory, cardiac and orthopaedic endpoints and life expectancy. Patients with <5% dystrophin expression ambulated longer (n=33) than patients without dystrophin (n=44), (p<0.001). Furthermore, patients with <5% dystrophin had longer survival (p<0.001), delayed decline in Left Ventricular Ejection Function [LVEF] <55% (p<0.001), delayed decline in vital capacity [CV] <50% (p<0.001) and in CV<20% (p<0.001), later start of non-invasive ventilation (p<0.001), tracheotomy (p<0.001) and arthrodesis (p<0.001). In conclusion, very low residual dystrophin expression is sufficient to shift the natural history of dystrophinopathy towards the milder Becker phenotype. These data suggest that restoration of low dystrophin levels might be sufficient to achieve long-term clinical benefit from novel therapeutic strategies. Results will be discussed in the light of the limited number of study subjects, time of observation and employed western blot methodology.

Genetic analysis of 1051 Chinese families with Duchenne/Becker Muscular Dystrophy

BackgroundDuchenne Muscular Dystrophy (DMD) is the most common muscle disease in children, and there are no effective therapies for DMD or Becker Muscular Dystrophy (BMD). Currently, targeted gene therapy treatments have emerged. As a result, genetic diagnosis is the basis of treatment. In addition, genetic and prenatal diagnosis significantly reduces their incidence rates. This study combines the application of multiplex ligation-dependent probe amplification technology (MLPA) and “next-generation” sequencing technology (NGS) as the most economical and efficient method of diagnosis. Therefore, in the diagnosis of DMD/BMD, patients’ MLPA data are first used to detect DMD gene deletions or duplications, and NGS and Sanger sequencing are then applied to exclude MLPA-negative samples. Meanwhile, polymerase chain reaction (PCR) is used to detect single exon deletions to exclude false-positives in MLPA caused by point mutations.MethodsIn this study, we recruited 1051 proband families of DMD from 2016 to 2018 and had access to information that could identify individual participants during or after data collection. Patients who were diagnosed with DMD were first tested by MLPA. MLPA results with single exon deletions were validated with PCR amplification and Sanger sequencing. The negative results of MLPA were further analysed with NGS and validated by Sanger sequencing. For novel missense mutations, phenotype-genotype correlations were analysed using PolyPhen2 and mutation taster. All methods were performed in accordance with the relevant guidelines and regulations.ResultsDMD mutations were identified in 1029 families (97.91%, 1029/1051). Large deletions, duplications, and small mutations accounted for 70.41% (740/1051), 8.28% (87/1051), and 19.12% (201/1051) of all cases, respectively. There were 205 small mutation types, 53 of which were novel. The rate of de novo mutations was 39.45% (187/474) and was higher in large duplications (49.53%, 157/317). Among 68 asymptomatic patients (< 3 years old) with unexplained persistent hyperCKaemia upon conventional physical examination, 63 were diagnosed as DMD/BMD according to genetic diagnosis.ConclusionOur results expand the spectrum of DMD mutations, which could contribute to the treatment of DMD/BMD and provide an effective diagnosis method. Thus, the combination of MLPA, NGS and Sanger sequencing is of great significance for family analysis, gene diagnosis and gene therapy.

Molecular Genetics Analysis of 70 Chinese Families With Muscular Dystrophy Using Multiplex Ligation-Dependent Probe Amplification and Next-Generation Sequencing.

Background: Muscular dystrophy (MD) includes multiple types, of which dystrophinopathies caused by dystrophin (DMD) mutations are the most common types in children. An accurate identification of the causative mutation at the genomic level is critical for genetic counseling of the family, and analysis of genotype–phenotype correlations, as well as a reference for the development of gene therapy. Methods: Totally, 70 Chinese families with suspected MD probands were enrolled in the study. The multiplex ligation-dependent probe amplification (MLPA) was first performed to screen large deletions/duplications of DMD exons in the patients, and then, next-generation sequencing (NGS) was carried out to detect small mutations in the MLPA-negative patients. Results: Totally, 62 mutations of DMD were found in 62 probands with DMD/BMD, and two compound heterozygous mutations in LAMA2 were identified in two probands with MDC1A (a type of congenital MD), indicating that the diagnostic yield was 91.4% by MLPA plus NGS for MD diagnosis in this cohort. Out of the mutations, 51 large mutations encompassing 47 (75.8%) deletions and four duplications (6.5%) were identified by MLPA; 11 small mutations including six (9.7%) nonsense, two (3.2%) small deletions, two splice-site mutations (3.2%), and one small insertion (1.6%) were found by NGS. Large mutations were found most frequently in the hotspot region between exons 45 and 55 (70.6%). Out of the 11 patients harboring point mutations in DMD, 8 were novel mutations. Additionally, one novel mutation in LAMA2 was identified. All the novel mutations were analyzed and predicted as pathogenic according to American College of Medical Genetics and Genomics (ACMG) guideline. Finally, 34 DMD, 4 BMD, 24 BMD/DMD, and 2 MDC1A were diagnosed in the cohort. Conclusion: Our data indicated that the MLPA plus NGS can be a comprehensive and effective tool for precision diagnosis and potential treatment of MD and is particularly necessary for the patients at very young age with only two clinical indicators (persistent hyperCKemia and typical myopathy performance on electromyogram) but no definite clinical manifestations.